HCFC1 variants in the proteolysis domain are associated with X‐linked idiopathic partial epilepsy: Exploring the underlying mechanism

Abstract Background HCFC1 encodes transcriptional co‐regulator HCF‐1, which undergoes an unusual proteolytic maturation at a centrally located proteolysis domain. HCFC1 variants were associated with X‐linked cobalamin metabolism disorders and mental retardation‐3. This study aimed to explore the role of HCFC1 variants in common epilepsy and the mechanism underlying phenotype heterogeneity. Methods Whole‐exome sequencing was performed in a cohort of 313 patients with idiopathic partial (focal) epilepsy. Functional studies determined the effects of the variants on the proteolytic maturation of HCF‐1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. Results We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability. Conclusion HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF‐1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub‐molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.


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of the variants on the proteolytic maturation of HCF-1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. Results: We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability.
Conclusion: HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF-1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub-molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.

K E Y W O R D S
cobalamin metabolism disorders, HCFC1 variant, molecular sub-regional effect, partial epilepsy, proteolysis dysfunction

INTRODUCTION
The host cell factor C1 gene (HCFC1, MIM *300019) is located at Xq28 and encodes HCF-1, which is ubiquitously expressed across the whole lifespan with predominant expression in the embryonic brain and associated with psychomotor development. 1,2 Mature HCF-1 is a heterodimeric complex formed by its own N-and C-terminal fragments after proteolysis. 3 As a transcriptional coregulator, HCF-1 plays critical roles in multiple biological processes, such as cell proliferation, migration and cell death. 4 In embryonic murine neural cells, over-expression of HCF-1 reduces hippocampal neuronal arborization and increases neurotoxicity, 2 while knockdown of HCF-1 results in expansion of neural progenitor cells and promotes axonal growth of post-mitotic neurons. 5 In mice, knockout Hcfc1 allele in the epiblast of male embryos leads to embryonic lethality. 6 In humans, HCFC1 variants have been associated with X-linked recessive cobalamin metabolism disorder that presented severe epileptic encephalopathy, intellectual disability, failure to thrive and even early death (designated CblX, MIM #309541) 7,8 and also have been reported in intellectual disability with/without epilepsy but without metabolic disorders. 2,9,10 It is suspected that HCFC1 variants were associated with common epilepsy and the relationships between cobalamin metabolism disorders, intellectual disability and epilepsy remain elusive. It is noted that mature HCF-1 complex possess a specific structure with distinct function of each domain. It is unknown whether the phenotype variations are associated with the functional domains.
In this study, trio-based whole exome sequencing (WES) was performed in a cohort of patients with epilepsies of unknown causes (idiopathic). Seven hemizygous HCFC1 variants were identified in 11 unrelated cases with idiopathic partial (focal) epilepsies without cobalamin disorder. Functional studies were performed to determine the effects of the variants on the proteolytic maturation of HCF-1, cell proliferation and expression of metabolism of cobalamin associated C gene (MMACHC, MIM *609831). We confirmed the role of HCFC1 variants in partial epilepsy in another cohort from multiple centers. The molecular sub-regional effects of HCFC1 variants were analysed.

Patients
A total of 463 unrelated cases of epilepsy without acquired causes were consecutively enrolled from the Epilepsy Center of the Second Affiliated Hospital of Guangzhou Medical University from 2012 to 2019. The cohort consisted of 313 cases with idiopathic partial epilepsies and 150 cases with idiopathic generalized epilepsies. Brain MRI (magnetic resonance imaging) was performed to exclude symptomatic epilepsy. Video-EEG (electroencephalogram) was performed and reviewed by two qualified electroencephalographers. Epileptic seizures and epilepsy were diagnosed and classified according to the criteria of the Commission on Classification and Terminology of the International League Against Epilepsy (1989, 2001, 2010 and 2017). As normal controls (in-house), 296 healthy Chinese volunteers were recruited as in our previous report. 11,12 To verify the role of HCFC1 variants in epilepsy, additional 80 cases with idiopathic partial epilepsy were collected from other seven hospitals in China.
This study received approval from the ethics committee of the Second Affiliated Hospital of Guangzhou Medical University, and adhered to the guidelines of the International Committee of Medical Journal Editors with regard to patient consent for research or participation. All participants and their parents provided written informed consents.

Expression construct and transfection
The coding sequences of human HCFC1 (NM_005334.2) and amino-terminal HA epitope tag were cloned into pcDNA3.1(+) expression vectors. Mutant constructs were generated using the ClonExpressII One Step Cloning Kit (Agilent Technologies) and transfections were conducted using Lipofectamine 2000 (Invitrogen). Primer sequences are available upon request. All variants were verified by direct sequencing.

HCF-1 proteolytic cleavage assay
To study HCF-1 proteolysis processing, full-length wildtype and mutant HCF-1 were expressed transiently in HEK293T cells and epitope-tagged amino-terminal polypeptides were subsequently detected by immunoblotting. Briefly, 48 h after transfection, approximately 80 µg of protein extracts were separated on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Blots were blocked with 5% skimmed milk and then probed with 1:1000 anti-HA tag antibody (Abcam, ab9110) at 4 • C overnight. After washes, the blots were incubated with the appropriate secondary antibody (Proteintech, SA00001-2) conjugated to horseradish peroxidase for 2 h. The immunodetection was performed using the chemiluminescent enhanced chemiluminescence reagents (Bio-Rad).

Liquid chromatography mass spectrometry identification
To identify the proteolytic products, the immunoprecipitation and mass spectrometry analysis were conducted.
Briefly, 48 h after transfection, HEK293T cells were lysed on ice in IP Lysis Buffer. After centrifugation, 1000 μg of supernatant was incubated with 10 μg anti-HA (Abcam, ab9110) and 1 µg normal mouse anti-IgG antibody (CST, 5415S) overnight at 4 • C, and then incubated with 20 μL of protein G/A magnetic beads for 4 h at 4 • C. After washes, the proteins were resuspended and then separated on a 7.5% SDS-PAGE stained with silver stain (Beyotime). The stained protein bands were excised and trypsinized for analyses with liquid chromatography (LC) in combination with electrospray ionization-tandem mass spectrometry (MS/MS) on a Triple time of flight (TOF) 5600+LC/MS system (AB SCIEX). The raw MS/MS data were analysed using ProteinPilot (version 4.5).

Quantitative real time polymerase chain reaction (qRT-PCR)
To study the effect of HCFC1 variants on cobalamin metabolism, we examined MMACHC expression. Total RNA was isolated from cultured HEK293T cells with HiPure Total RNA Kits (Magen), and cDNA was generated using Toyobo's ReverTra Ace qPCR RT Kit (Toyobo). Quantitative PCR analysis (200 ng) was performed using Thunderbird SYBR qPCR Mix (Toyobo) in a LightCycler 480 (Roche) system. The relative gene expression was calculated using the 2-∆∆C T method with GAPDH as an internal control. Primers for GAPDH, HCFC1 and MMACHC are available upon request.

Immunofluorescence
To observe the sub-localization of wild-type and mutant HCF-1, we employed immunofluorescent staining and confocal laser scanning microscopy. Forty-eight hours after transfection, the fixed and blocked cells were incubated overnight at 4 • C with the 1:500 primary antibodies (Abcam, ab9110) and then incubated with appropriate Alexa-488-conjugated secondary antibodies (Abcam, ab150077) for 2 h. After rinsing, cells were counterstained with DAPI. Fluorescence was visualized by confocal microscopy on a Leica TCS, SP8 microscope.

Cell proliferation assay
To examine the effect of HCFC1 variants on cell proliferation, the growth rate of HEK293T cells was determined using the Cell Counting Kit-8 (CCK-8; Beyotime). Briefly, HEK293T cells or transfected HKE293T cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and cultured for 48 h at 37 • C, and treated with 10 μl/well CCK-8 solution (5 mg/ml) for 4 h. The absorbance was measured at 450 nm using a microplate reader (Tecan). Six parallel wells were set in each group, and the mean value was obtained.

Statistical analysis
The Student's t-test was used to compare two independent samples, and one-way ANOVA analysis was applied to compare multiple samples. Two-tailed Fisher's exact test was used to compare allele frequencies between groups. Statistical analyses were performed with R statistical software (v4.0.3) and SPSS statistics 26.0. The p value < .05 was considered statistically significant.

Identification of HCFC1 variants
Seven hemizygous HCFC1 variants were identified in 11 unrelated patients in our trio-based cohort of 313 cases with idiopathic partial epilepsies. The hemizygous variants included c.3277_3285delACCGCCACC/ p.Thr1093_Thr1095del, c.3356C > T/p.Thr1119Ile, c.3757C > T/p.Arg1253Cys, c.3790G > A/p.Gly1264Ser, c.3845C > T/p. Ser1282Leu, c.4217C > T/p.Ala1406Val and c.4384G > A/p. Asp1462Asn. The p.Thr1119Ile was recurrently detected in five cases ( Figure 1 and Table 1). All the 11 patients with HCFC1 variants were boys. In contrast, the female carriers with heterozygous variants were asymptomatic ( Figure 1). It is consistent with the X-linked recessive inheritance pattern, as the previously reported cases with HCFC1 variants. 2,5,7 No hemizygous or homozygous HCFC1 variants were identified in the 150 patients with idiopathic generalized epilepsies.
The c.3790G > A/p.Gly1264Ser and c.4384G > A/p.Asp 1462Asn were not present in any public databases, including the 1000 Genomes Project, Exome Aggregation Consortium, and gnomAD. The other five variants were observed with a very low allele frequency in the control populations of gnomAD as hemizygotes ( Table 2). Homozygotes of these variants were not detected in general populations. All the variants were absent from our inhouse 296 normal controls. We compared the frequencies of these hemizygous variant alleles in the present cohort with that in control populations of gnomAD (Table 2). In this cohort, 11 hemizygous mutant alleles in a total of 438 alleles (.025114, 188 male and 125 female cases) were detected, which was significantly higher than that in the TA B L E 1 Clinical and genetic features of the patients with HCFC1 variants in our cohort. controls of the East Asian population (.002119, 14/6606, p = 9.6 × 10 −8 ) and that in the controls of all populations in gnomAD (.000226, 18/76492, p = 2.2 × 10 −16 ). None of the 11 cases had other pathogenic or likely pathogenic variants in genes known to be associated with seizures. 14 The c.3277_3285delACCGCCACC/p.Thr1093_Thr1095del was potentially deleterious, yielding a 3-amino-acid deletion of the protein HCF-1. The other six missense variants were predicted to be damaging by multiple predictors (Table S1). Three of the missense variants (p.Thr1119Ile, p.Ala1406Val and p.Asp1462Asn) changed their hydrogen bonds with surrounding residues ( Figure S1B). We further studied the effects of HCFC1 variants on the function of HCF-1 experimentally.

Effect on proteolytic cleavage
HCF-1 precursor consists of a kelch domain, a basic domain, a proteolysis domain, an acidic domain, three fibronectin 3 domains (Fn3) and a nuclear localization signal (NLS) domain (Figure 2A). The proteolysis domain contains six centrally located and highly conserved 26amino-acid repeats, named HCF-1 PRO repeats 1−6. Each HCF-1 PRO repeat contains a cleavage region (N6-H12) and a threonine region (T14-T24) that is a binding site of Olinked β-N-acetylglucosamine transferase (OGT). 15,16 After binding, OGT will O-GlcNAcylate the HCF-1 N subunit and directly cleave the HCF-1 PRO repeat at E10 cleavage site in a stochastic manner. 15,16 Six of the variants identified in this cohort, p.Thr1093_Thr1095del, p.Thr1119Ile, p.Arg1253Cys, p.Gly1264Ser, p.Ser1282Leu and p.Ala1406Val were located in the proteolysis region ( Figure 2A). The variant p.Thr1093_Thr1095del yielded a deletion containing the essential binding threonine (T22) of the repeat 2, and potentially disrupting the binding with OGT. The variant p.Thr1119Ile substituted the essential binding threonine (T19) of the repeat 3, potentially leading to loss a binding point with OGT. The other four variants within the proteolysis region were located between the repeats. One of the variants, p.Asp1462Asn was located in the acidic domain.
To examine the effect of mutants on the cleavage, a positive control (c.3242A > C/p.Glu1081Ala) and a negative control (c.6046C > T/p.Arg2016Trp) were set. The mutation p.Glu1081Ala, which lies in the E10 cleavage site of the repeat 2, has previously been verified to block cleavage. 3,15,16 The variant p.Arg2016Trp, which is located in the C-terminal NLS domain and shown to disrupt the nuclear localization in the previous study, 5 was theorized to have no effect on cleavage.
The wild-type HCF-1 protein generated at least six amino-terminal HA epitope-tagged polypeptides labeled a-f ( Figure 2B), similar to the previous studies. 3,17 The relative sizes of these six bands a to f potentially represent amino-terminal cleavage products of repeats 1 to 6, respectively. The largest size of a ∼300 kD polypeptide (the band g) corresponds to the full-length translation product HCF 300. As shown in Figure 2B, the positive control p.Glu1081Ala resulted in the absence of the second and third shorter polypeptides (band b and c) compared to the wild-type HCF-1. Variants p.Thr1093_Thr1095del and p.Thr1191Ile, which were located in the canonical proteolytic sites in the threonine regions of the repeat 2 and repeat 3, respectively, resulted in the absence of the second (band b) and third (band c) smaller polypeptides, respectively. The variant p.Arg1253Cys was located between the repeats and near the center of the proteolysis domain and did not affect the formation of the first three shorter HCF-1 polypeptides; but surprisingly, it resulted in a new major epitope-tagged polypeptide of ∼140 kD (band n) with loss of the next three cleaved fragments (band d to f) and the largest protein HCF 300 (band g). Three variants located between the repeats (p.Gly1264Ser, p.Ser1282Leu, and p.Ala1406Val), and p.Asp1462Asn in the acidic domain, did not disrupt the proteolytic processing compared to the wild-type ( Figure 2B and Figure S2).
The new proteolytic product of mutant p.Arg1253Cys (band n, indicated in the red box in Figure 2B) was excised and analysed by LC-MS/MS. As shown in Figure 2C, the band n contained 31 unique HCF-1 peptides (Table S2, Figure S3.1-S3.31), which covered the amino acids before residue p.Arg1253 (indicated in red in Figure 2D). The last peptide sequence, SPAFVQLAPLSSK from amino acids 1205 to 1217, was 36 amino acids away from the residue p.Arg1253, suggesting the cleavage point was between the amino acids 1217 to 1253.

Effect on cell proliferation
HCF-1 potentially inhibits cell proliferation via regulating cell-cycle progression. 18,19 In this study, the growth rate of HEK293T cells was measured by CCK-8 assay after 48 h of growth. The overexpressed wild-type HCFC1 resulted in a 50% decrease in growth compared with the blank control cultures (transfected with an empty expression vector), showing a distinct inhibition on cell proliferation ( Figure 3A), consistent with the previous report. 5 Comparing with the wild-type HCFC1, all the nine variants caused increases in cell number with a range from 18% to 73%, indicating loss of growth suppression ( Figure 3A). The increase was statistically significant in eight of nine variants. The p.Asp1462Asn mutant, which was located in  the acidic domain, resulted in increases in growth of 18%, showing a modest, but not significant difference from wildtype HCF-1. The data suggest these variants may cause loss of function in inhibition of cell proliferation of various degrees.

Effect on MMACHC expression
MMACHC, one of downstream target genes regulated by HCFC1, encodes a cobalamin transport protein with enzymatic capabilities. Previous studies have revealed that HCFC1 variants reduced MMACHC expression in fibroblasts derived from patients with cobalamin metabolism disorders. 4,7,20 This study utilized HEK293T cells and over-expressed wild-type or mutant HCF-1 and analysed the expression of MMACHC by qPCR as previously described. 5 As shown in Figure 3B, the relative expression of MMACHC in the nine HEK293T cell lines with HCFC1 variants were similar to that with wild-type HCFC1, suggesting that these nine HCFC1 variants did not affect the expression of MMACHC.

HCFC1 variants on nuclear localization
As shown in Figure 3C, the wild-type HCF-1 was predominantly distributed in the nucleus. In contrast, the mutant p.Arg2016Trp, in which the mutation was located in the NSL and affected the nuclear localization, was enriched in the cytoplasm, as that in the previous study. 5 The p.Arg1253Cys mutant also failed to localize to the nucleus and was detected predominantly in the cytoplasm, suggesting a loss of ability in nuclear localization. The other seven HCFC1 variants were expressed mainly in the cell nuclei, akin to the wild type.

Clinical features of patients with HCFC1 variants
The clinical characteristics of the 11 cases with HCFC1 variants are summarized in Table 1. The seizure onset age ranged from 8 months to 24 years old, with a median onset age of 3 years. All the patients with missense variants started seizures in infancy or childhood, while the patient with in-frame deletion had late-onset age of 24 years old. All the patients had focal seizures or focally originating tonic-clonic seizures characterized by shifting or bilateral focal discharges (Figure 4). Among them, five cases were diagnosed as Rolandic epilepsy, one case as occipital lobe epilepsy, three cases as partial epilepsies without specific characteristic, and two cases of partial epilepsy with additional generalized seizures (case 8 and 11; Figure 1 and Table 1). Frequent daily seizures were observed in case 7 with p.Arg1253Cys. He had an early-onset age of 8 months and suffered from focal to bilateral tonic-clonic seizures with a frequency of up to five times per day, which lasted for 3 years and then followed by complex partial seizures. The seizures in case 7 seem more severe than that in other cases. All the patients achieved seizure-free with antiepileptic drugs.
Four cases, including two cases with p.Thr1119Ile (case 5 and 6), case 7 with p.Arg1253Cys, and case 11 with p.Asp1462Asn, displayed mild intellectual disability with difficulties in communication ( Table 1). The motor development of all cases was normal. Brain MRI was normal in all cases.
Blood biochemical investigations of the cobalamin metabolism, including plasma vitamin B12, folate, and homocysteine levels, were conducted in eight of the 11 cases. The metabolic evaluations in these eight patients were essentially normal (Table 1).

HCFC1 variants in validation cohort
To confirm the association between HCFC1 variants and epilepsy, we collected additional 80 cases with partial epilepsy but without acquired causes from other clinical centers, including 31 trios and 49 singletons. Two variants (p.Thr1119Ile and p.Arg1253Cys) were recurrently identified in three additional patients each, including one homozygous p.Thr1119Ile and one homozygous p.Arg1253Cys (Table S3) The clinical and genetic features of the 13 patients with HCFC1 variants from the validation cohort were summarized in Table S3. The onset age of seizures ranged from the first day to 9 years old, with a median onset age of 2 years. All of the cases had focal seizures or focal to bilateral tonic-clonic seizures that can be controlled by monotherapy or polytherapy of antiepileptic drugs. The EEG recordings showed focal discharges. The two boys with p.Arg1253Cys both had very early-onset age, frequent daily seizures and intellectual disability, sharing similar symptoms with case 7 with p.Arg1253Cys in our cohort. One boy with p.Ile632Val and one with p.His1235Gln had mild intellectual disability. All the cases had normal motor development.

Molecular sub-regional implications of HCFC1 variants
Previous studies have reported HCFC1 variants in X-linked cobalamin disorders 7,8 (CblX) and nonsyndromic intellectual disability without cobalamin abnormalities. 2,5,10,21 The present study identified novel HCFC1 variants as a potential cause of benign partial epilepsy without cobalamin disorder. To understand the mechanism underlying phenotypic variations, we analysed genotype-phenotype associations in all HCFC1 variants with detailed phenotypes. Publications on HCFC1 variants were retrieved from the PubMed database till Oct 2022. To date, 48 variants, including 44 missense variants, two splicesite variants, a small deletion, and a 5′-UTR variant, were identified in 98 individuals from 75 families. Their molecular locations and clinical data were listed in Table S4.
Six missense variants were associated with cobalamin disorders, which were reported in 18 unrelated cases. All of the CblX-associated variants were located within the K1 and K2 motifs of the kelch domain ( Figure 5A).  . (B) A proposed model of OGT-induced HCF-1 proteolytic maturation and the potential molecular sub-regional effects of HCF-1. The HCF-1 precursor undergoes an unusual proteolytic maturation that is mediated by OGT. Cleavage occurs at the six centrally located HCF-1 PRO repeats in a stochastic manner. The proteolytic HCF-1 N and HCF-1 C terminal fragments are noncovalently associated through two matched pairs of self-association sequences (SAS1N-SAS1C and SAS2N-SAS2C). Variants within the proteolysis domain, which does not exist in the mature HCF-1 complex, were associated with benign epilepsy. Variants in the kelch domain were associated with cobalamin disorders, and those in the self-association complex were mainly associated with intellectual disability without cobalamin disorder.
Twenty-one missense variants were previously reported to be associated with intellectual disability without cobalamin disorders. The variants were located in regions from the K4 motif to the C terminus, of which majority (13/21, 61.9%) clustered in the basic and acidic domains, and only one in the proteolysis domain. In contrast, majority (11/13, 84.6%) of the variants identified in this study were located in the proteolysis domain and were associated with partial epilepsy without cobalamin disorder. The other two variants were located in the basic and acidic domains, respectively. The data suggested a molecular sub-regional effect of HCFC1 variants.

DISCUSSION
The present study identified seven hemizygous HCFC1 variants in 11 unrelated cases from a cohort of idiopathic partial epilepsies, and confirmed the finding in another cohort with additional 13 unrelated cases. None of patients had cobalamin disorders. Majority of the variants (11/13, 84.6%, in 91.7% cases) were located in the proteolysis domain. Functional studies demonstrated that the variants in the proteolysis domain potentially disrupted the proteolytic maturation of HCF-1 and the inhibition of cell proliferation but did not affect MMACHC expression that was associated with cobalamin metabolism. Further genotypephenotype analysis demonstrated that variants within the proteolysis domain were associated with common partial epilepsy without cobalamin disorders, whereas those in the kelch domain were associated with cobalamin disorders featured by fatal epileptic encephalopathy, and those in the basic and acidic domains were mainly associated with intellectual disability without cobalamin disorders. The present study suggested that HCFC1 was potentially a candidate pathogenic gene of common epilepsy with distinct mechanism of proteolysis dysfunction. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype outcome and pathogenic mechanism, implied a clinical significance in early diagnosis and management. Idiopathic partial (focal) epilepsy is a common group of self-limited childhood epilepsies with infrequent focal seizures, benign course, and good prognosis, typically Rolandic epilepsy, that affects .2% of the population with an incidence of 10−20/100,000 children. [22][23][24] Generally, the minor allele frequency (MAF) of genetic variants in general populations depends on phenotype prevalence 25 that is related to phenotype severity. Variants that are associated with common diseases or mild phenotypes could be prevalent with low MAF in general populations, in contrast to those with rare diseases or severe phenotypes that usually are absent in general populations. In the present study, five of the variants within the proteolysis domain presented low frequencies (MAF < .0005) in general populations of gnomAD (Table 2), similar to some variants in genes associated with common partial epilepsy, such as those in GRIN2A, 26 DEPDC5 27 and UNC13B. 12 Incomplete penetrance is common in genetic disorders and challenges evaluation of the pathogenicity of variants. Functional studies were further performed to validate the damage effect of these variants. The HCFC1 variants within the proteolysis domain disrupted the proteolytic processing with loss of growth suppression but did not affect MMACHC expression that was associated with cobalamin disorder and severe epileptic encephalopathy. The plain and mild functional consequences of the variants were consistent with the mild clinical phenotype with incomplete penetrance and low MAF in general populations.
The precursor HCF-1 comprised of several conserved protein domains and undergoes an unusual proteolytic maturation that is mediated by OGT ( Figure 5). 15,16 Cleavage occurs at the six centrally located HCF-1 PRO repeats in a stochastic manner. The proteolytic HCF-1 N and HCF-1 C terminal fragments are non-covalently associated with each other through two matched pairs of self-association sequences (SAS1N-SAS1C and SAS2N-SAS2C), 28 forming a mature HCF-1 complex ( Figure 5B). As a transcriptional coregulator, HCF-1 functions as a molecular scaffold to link sequence-specific transcription factors with enzymes capable of altering posttranslational modifications, forming a critical component of transcriptional regulatory network governing cell proliferation, cell-cycle progression and cell viability. 29 Proteolysis domain, which does not exist in the mature HCF-1 complex, conducts proteolytic process that is necessary to separate and ensure the HCF-1 N and HCF-1 C functions in cell proliferation. 18 The HCF-1 N subunit promotes passage through the G1 phase of cell growth, and the HCF-1 C subunit regulates proper exit from mitosis. 18,19,30,31 Previous experiments have demonstrated that variants, including large/small deletions of the repeats and point mutations in the cleavage and threonine regions (e.g., p.Glu1081Ala), disrupted the cleavage process. 3,15,16 However, the variant-associated clinical phenotypes have not been reported previously. The present study identified 11 variants in the proteolysis domain in 22 unrelated cases with partial epilepsy. The functional studies demonstrated that the variants in the canonical proteolytic sites in the threonine regions (p.Thr1093_Thr1095del and p.Thr1119Ile) disrupted the cleavage. The variant p.Arg1253Cys, located near the center of the proteolysis domain, resulted in obviously abnormal proteolytic fragments ( Figure 2B). The three cleavage-affected variants significantly affected the subsequent cell proliferation ( Figure 3A). The other three variants between the repeats (p.Gly1264Ser, p.Ser1282Leu and p.Ala1406Val) did not cause visibly abnormal cleavage products but significantly caused loss of function in inhibition of cell proliferation ( Figure 3A). These findings suggested that the proteolysis dysfunction of HCF-1 and loss of growth suppression is potentially a novel pathogenic mechanism for epilepsy.
The mutant p.Arg1253Cys, which was located between the repeats, resulted in a distinct abnormal cleavage, suggesting the mutations in the cryptic regions beyond the canonical sites were also potentially affecting proteolytic process. Furthermore, mutant p.Arg1253Cys failed to localize on nucleus. The failure in nuclear localization was supposed to be caused by the loss of NLS in the HCF-1 C subunit that may not be self-associated with the new polypeptide after proteolysis. Clinically, the three cases with p.Arg1253Cys all presented severer manifestations with early-onset age before 8 months and frequent daily seizures, potentially explained by the profound functional alterations of p.Arg1253Cys.
Previously reported variants clustered in the basic domain and the acidic domain were associated with intellectual disability without cobalamin disorder. In the present study, the patients with variants in the basic and acidic domains also had intellectual disability. Whereas, only six (27.3%) of the 22 patients with variants in the proteolysis domain had intellectual disability. The basic and acidic domains are core regions of SAS2N and SAS2C, which interact with each other to form a second association complex for the heterodimeric HCF-1 ( Figure 5B). The present study demonstrated that the variant (p.Asp1462Asn) in the acidic domain resulted in loss of inhibition of cell proliferation, consistent with the previous studies on variants in the basic and acidic domains. 5 Cell proliferation leading to seizures is a common phenomenon. For example, variants in mTOR signaling pathway genes (e.g., TSC1, TSC2, MTOR, DEPDC5, NPRL2 and NPRL3), 32 which are involved in growth and cell proliferation, cause a spectrum of partial epilepsy syndromes with or without visible cortical structural abnormalities. Besides the dysfunction in regulating cell proliferation, variants in the basic and acidic domains potentially caused elongated axonal length of hippocampal neurons. 5 Recently, a knock-in zebrafish model harboring mutation in the hcfc1a gene (the hcfc1a co60/+ ), one ortholog of HCFC1, increased neural precursor cells and expression of neuronal and glial markers, and subsequently led to abnormal swim patterns without speed deficits. 33 These findings suggested the role of the basic and acidic domains in the neurodevelopment.
None of the patients with variants in the present study had clinical manifestation of cobalamin abnormalities. The functional study showed that variants in the proteolysis domain did not affect the expression of MMACHC.
Further genotype-phenotype analysis demonstrated that the variants associated with cobalamin disorders were all located within the K1 and K2 motifs of the kelch domain. The kelch domain of HCF-1 forms an antiparallel β-propeller structure that exerts protein-protein interactions with transcription promoters. 34,35 Through binding with Thanatos-associated protein 11 (THAP11), 29,36 HCF-1 regulates expression of MMACHC, 4,7 which encodes a critical enzyme of the cobalamin pathway. Previous studies have showed that the CblX-associated variants, which were within or adjacent to the β strands, 35 severely reduced MMACHC expression in skin fibroblasts of patients with cobalamin disorders 7 and in "cblX" mice with Hcfc1 A115V/Y . 37 In contrast, a K4-located variant p.Ser225Asn, which was distant from β strands, 35 showed a slight impact on the MMACHC expression 5 and was not associated with cobalamin deficiency 2 and there was a quantitative correlation between the degree of phenotypic penetrance and the MMACHC expression modulated by HCFC1. 4 In conclusion, the present study indicated that HCFC1 was potentially a candidate gene for common idiopathic partial epilepsy with distinct underlying mechanism of the proteolysis dysfunction and loss of growth suppression. The HCF-1 domains played distinct functional roles and were associated with different clinical phenotypes, highlighting the sub-molecular mechanism underlying phenotype heterogeneity. Disclosing the sub-molecular effects and establishment of HCFC1-epilepsy association will help the early diagnosis and management of patients with HCFC1 variants.

A C K N O W L E D G E M E N T S
The authors thank the family and physicians for participation in our study. They are grateful to the He Shanheng Charity Foundation for contributing to the development of this institute. This work was funded by the National Natural Science Foundation of China (grant numbers: 81971216 to HN, 82171439 to SYW and 82071548 to TB), Guangdong Basic and Applied Basic Research Foundation (grant numbers: 2020A1515011048 to HN, 2021A1515110792 to LWB, 2021A1515111064 to WJ and 2021A1515010986 to SYW), the Science and Technology Project of Guangdong Province (grant number: 2017B030314159 to LWP), Science and Technology Project of Guangzhou (grant numbers: 201904010292 to HN and 201904020028 to LWP). The funders had no role in study design, data collection and analysis and decision to publish or preparation of the manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.